1. Field of the Invention
The present invention relates to a method for preparing heterodimeric analogs of cysteine knot proteins. More specifically, the invention relates to a method for forming a subunit combination of a cysteine knot protein having an xcex1-subunit and a xcex2-subunit to prepare a heterodimeric protein analog which comprises the steps of (a) attaching a dimerization domain to the amino termini of both an xcex1-subunit and a xcex2-subunit of a cysteine knot protein; and (b) dimerizing the xcex1-subunit and xcex2-subunit to form a heterodimeric protein analog. In another embodiment, the invention relates to a method for forming a subunit combination of a cysteine knot protein having an xcex1-subunit and a xcex2-subunit to prepare a heterodimeric protein analog which comprises the steps of (a) attaching a dimerization domain to the amino terminus of an xcex1-subunit and the carboxy terminus of a xcex2-subunit of a cysteine knot protein; and (b) dimerizing the xcex1-subunit and xcex2-subunit to form a heterodimeric protein analog.
2. Description of the Background
The disclosures referred to herein to illustrate the background of the invention and to provide additional detail with respect to its practice are incorporated herein by reference and, for convenience, are numerically referenced in the following text and respectively grouped in the appended bibliography.
The glycoprotein hormone family (1-3) consists of three xcex1, xcex2 heterodimeric glycoproteins found in the anterior pituitary gland where they are made and includes luteinizing hormone (LH), follicle stimulating hormone (FSH), and thyroid stimulating hormone (TSH). These hormones are found in most, if not all vertebrates. In some species, a glycoprotein hormone structurally similar to LH is found in the placenta wherein it is synthesized. The human placental hormone is known as human chorionic gonadotropin (hCG). In primates, significant quantities of all the hormones are also found as excretion products in urine. Urine from pregnant women serves as a convenient source of hCG. After menopause, when the secretion of LH and FSH from the anterior pituitary is greatly increased, significant quantities of LH and FSH are found in the urine and are termed human menopausal gonadotropins (hMG). Urine from menopausal women serves as an important source of LH and FSH activities. Urinary hormones (hCG, hMG, hFSH) and recombinant hormones have important clinical and commercial uses.
Gonadotropins such as CG, LH, and FSH play a major role in the reproductive process (4) while the structurally related hormone, TSH, is important for thyroid function. In women, FSH plays a crucial role in the development of follicles that can be ovulated, primarily through its influence on granulosa cells. LH synergizes with FSH and is normally essential for processes of ovulation, luteinization, and luteal function. Nonetheless, high LH levels can reduce fertility and are thought partly responsible for the loss of fertility associated with polycystic ovarian disease. hCG is important for maintenance of pregnancy and its early neutralization leads to infertility. In males LH is required for puberty and, in its absence, there is a failure to acquire the sexual attributes and fertility of an adult. The biological and clinical activities of these hormones are reviewed extensively in several textbooks including those by Yen and Jaffe (4), Adashi, Rock, and Rosenwaks (5), and DeGroot (6).
Both hCG and LH bind to luteinizing hormone receptors (LHR). In the testis, LHR are found primarily in the Leydig cells. In the ovary, LHR are found primarily in thecal cells, FSH-stimulated granulosa cells, and luteal cells. The major role of LH is to stimulate the formation of steroid hormones including the androgens testosterone and androstenedione (Leydig and thecal cells) and progesterone (FSH-stimulated granulosa, thecal, and luteal cells). LH also causes ovulation of mature follicles. While hCG is normally produced only by the placenta during pregnancy, due to its high affinity for LH receptors, the ease with which it can be purified from urine, and its long biological half-life, hCG has been widely used as a substitute for LH. Important clinical uses for hCG include stimulation of fertility in males and induction of ovulation in females.
FSH binds to FSH receptors (FSHR) located primarily in the Sertoli cells of the testis and the granulosa cells of the ovaries. The primary roles of FSH are to stimulate the conversion of androgens to estrogens, to promote the synthesis of inhibin and activin, to promote the development of Sertoli and granulosa cells, and to stimulate gamete maturation. The effect of FSH on granulosa cells leads to follicular maturation, a process during which the oocyte is prepared for ovulation and in which the granulosa cells acquire the ability to respond to LH. Follicle maturation is essential for the ability of LH to induce ovulation.
The differences in the effects of FSH and LH and the complex endocrine interactions between the two hormones cause them to have synergistic effects. For example, normal estrogen production is due to the effect of LH on androgen formation and the influence of FSH on the conversion of androgens to estradiol. Estrogens can inhibit the secretion of FSH and potentiate the secretion of LH. The ability of androgens to be converted to estrogens in non-ovarian tissues can disrupt this complex feedback interaction between steroidogenesis and the secretion of FSH and LH. For this reason, the ratio of LH/FSH activity as well as the absolute hormone levels in blood are important for reproductive functions such as ovulation of the proper number of oocytes during the menstrual and estrus cycles. Other hormones including activin and inhibin can exert an influence on this process, primarily through their influence on FSH secretion from the pituitary gland and their influence on the ovarian response to FSH.
TSH is produced in the anterior pituitary gland and its major function is to regulate the activity of the thyroid gland, causing it to synthesize and release thyroxin. Circulating levels of TSH and thyroxin are usually regulated by a negative feedback mechanism. Increases in TSH secretion usually lead to increased thyroxin synthesis and secretion by the thyroid. As thyroxin levels increase, the secretion of TSH is decreased. In this way there is a balance between the level of TSH and thyroid hormone. High levels of TSH can also stimulate the thyroid gland to remove iodine from circulation. Clinically, TSH can be used to promote the uptake of radioactive iodine and death of the thyroid cells. This form of thyroidectomy has been used to remove hyperactive thyroid tissues.
Hormones with FSH and LH activities are routinely used in the treatment of human infertility, a problem experienced by approximately 10-15% of all couples (7,8). A major cause of female infertility is polycystic ovarian disease or syndrome, a condition in which endogenous LH levels often appear to be elevated. In principle, infertility caused by inappropriately high LH activity could be suppressed by administration of an inhibitory hormone analog that competed with LH for binding to LHR. It has been known for many years (9,10) that it is possible to prepare analogs of hCG that act as LH antagonists by removing all or part of the oligosaccharides from the hormone. While it is possible to remove most of the oligosaccharides using endonucleases or exonucleases, in practice, it is not practical to remove all of them without denaturing the hormones. The remaining sugars can serve as substrates for enzymes and other factors that can hasten removal of the proteins from circulation (11-13). One potential means of avoiding this problem is to prepare analogs that have been genetically deglycosylated (i.e., by replacing or deleting amino acids in the signals needed for N-linked glycosylation). These signals have the amino acid sequence Asn-Xaa-Ser/Thr where Asn is asparagine, Xaa is any amino acid except proline, and Ser/Thr are serine or threonine. To disrupt glycosylation, Asn can be changed to any other amino acid, Xaa can be changed to proline and/or Ser or Thr can be changed to any other amino acids.
Using genetic deglycosylation, it has been shown that the oligosaccharide from the hCG xcex1-subunit at Asn52 has the greatest influence on signal transduction (10). Removal of this oligosaccharide leads to a substantial loss in hormone efficacy and enables the preparation of a partial agonist that can partially inhibit the response to hCG. However, because the other hormone oligosaccharides also influence signal transduction, preparation of the most potent antagonists requires that other N-linked amino acids, particularly those on the xcex1-subunit, be removed from the hormone (10). Unfortunately, removal of the xcex1-subunit oligosaccharide at Asn52 reduces the abilities of the xcex1- and xcex2-subunits to combine (10,14-16). While small amounts of heterodimer do form and can be studied in a laboratory setting (10), preparation of larger quantities needed for potential therapeutic uses is impractical. Methods for improving the production of deglycosylated glycoprotein hormone analogs are desirable and, as described later, one such method involves addition of dimerization sequences to the hormone subunits.
Hormone analogs that have prolonged half-lives or universal activities also have potential important uses. It is well known that the half-lives of the subunits is significantly shorter than that of the heterodimers [reviewed in Moyle and Campbell (2)]. Because dimerization domains can potentiate formation of heterodimers, they can also reduce the rate of hormone dissociation and influence circulation time. Hormone analogs that serve as immunogens are also potentially important. Dimerization domains can contain high immunogenic amino acid sequences and therefore increase the immunogenicity of the analogs.
The structures of the glycoprotein hormones have been studied for many years and the relative roles of the hormone subunits in receptor binding specificity are well-known (1). Glycoprotein hormones share a common xcex1-subunit and differ in their hormone-specific xcex2-subunits. The latter determine the biological and immunological properties of each hormone. Substitution of the xcex1-subunit of any one hormone for that of another does not change the receptor binding properties of the new hormone. Substitution of the xcex2-subunit is accompanied by a change in receptor binding specificity. Thus, when FSH xcex2-subunit is substituted for the LH xcex2-subunit, the recombined hormone acquires the properties of FSH and loses properties characteristic of LH. The sequences of many hormone subunits were determined several years ago and have been confirmed by their genomic and cDNA sequences (17-21).
The crystal structure of hCG determined in two laboratories (22,23) showed that each subunit had the overall topology characteristic of cysteine knot proteins (24). Each subunit is divided into three large loops by disulfide bonds that create the cysteine knot. Since the relative positions of the cysteines in all the glycoprotein hormones are very similar, it is nearly certain that the xcex2-subunits of LH, FSH, and TSH will also have a cysteine knot architecture. The xcex2-subunit differs from the xcex1-subunit in one important aspect, namely the presence of an additional sequence of approximately twenty amino acids that is attached to the C-terminal cysteine of the cysteine knot. In the xcex2-subunits of hLH, hCG, hFSH, and hTSH, the seatbelt corresponds to amino acid residues Gly91-Cys110, Ala91-Cys110, Gly85-Cys104, and Gly86-Cys105, respectively. This sequence was termed the seatbelt (22) because it is wrapped around the xcex1-subunit and forms a disulfide bond with a cysteine in xcex2-subunit loop 1 to stabilize the heterodimer. As reviewed by Ruddon and colleagues, the cysteine knot that latches the seatbelt appears to be one of the final steps in xcex2-subunit folding and appears to occur after the two subunits have combined (25).
Several attempts have been made to identify portions of the xcex1- and xcex2-subunits of the hormones that are responsible for their unique biological properties. Earlier studies were based on chemical modifications of the hormones (1). Modifications were described that reduced the biological activities of the hormones. Due to the complexity of the hormones, this approach was usually unable to identify single amino acid residues that were involved in receptor binding or binding specificity. In an attempt to simplify the problem of identifying residues involved in receptor binding, some investigators prepared synthetic peptides corresponding to partial sequences of the xcex1- and xcex2-subunits and monitored their abilities to inhibit binding of 125I-hCG and 125I-hFSH to LH and FSH receptors. Synthetic peptides corresponding to amino acid residues of hCG xcex2-subunit 38-57 or hFSH xcex2-subunit 31-52 appear to have higher abilities than most other peptides in these assays (26-29). However, they have extremely low affinities for the receptors, an observation that precludes their practical use.
A breakthrough in the ability to make and characterize glycoprotein hormone analogs came in 1985 when genetically engineered mammalian cells were first shown to express biologically active hCG heterodimers (30). Since that time several laboratories have used mammalian cells to express glycoprotein hormone analogs that are capable of binding to receptors and inducing or inhibiting signal transduction (14,31-37). These analogs appear to be glycosylated similarly to the naturally occurring hormones. In these procedures one introduces a xe2x80x9cgenexe2x80x9d that encodes the desired amino acid sequences into mammalian cells downstream of a promoter. Construction of these genes is a standard recombinant DNA procedure. By changing, adding and/or deleting codons in the hormone xcex1- or xcex2-subunit cDNAs or genomic DNA fragments using standard procedures, it is possible to build gene constructs that encode the desired analogs (38,39). When these constructs are transfected into mammalian cells by calcium phosphate precipitation, electroporation, or other standard protocols (38-40), they direct the synthesis of the hormone analogs and their secretion into the culture media. These media can be assayed for the presence of immunological or biological activity (31,32,41). Unfortunately, not all such constructs yield practical amounts of heterodimers. This is especially evident with hormones that lack one or more glycosylation signals.
Using mammalian cell expression systems to make hormone analogs, Campbell, et al. (31) showed that it was possible to switch the receptor binding activity of hCG. They engineered an analog that was chemically and immunologically more similar to hCG than hFSH, but that bound to FSH receptors much better than hCG and had only slightly higher affinity for LH receptors than FSH. Subsequent reports (33) showed that it was possible to prepare analogs of hCG that had a high affinity for both LH and FSH receptors. This was accomplished by replacing hCG seatbelt residues 101-109 with their hFSH xcex2-subunit counterparts (i.e., hFSH xcex2-subunit residues 95-103). These hCG analogs (31,33) elicit signal transduction at either LH and/or FSH receptors. This demonstrated that the seatbelt of the xcex2-subunit had a major influence on receptor binding specificity. It is anticipated that removing the oligosaccharides from analogs in which the specificity is modified by substitutions in the seatbelt will reduce their efficacy and cause them to become partial agonists and/or antagonists. Their ability to bind to receptors requires that the xcex2-subunits of these analogs combine with the xcex1-subunit to form heterodimers. The method described here will be useful for expressing these analogs as heterodimers and represents a significant advance in heterodimer preparation.
Slaughter et al. (42) showed that an interaction between the N-terminal portion of hCG xcex2-subunit and the seatbelt had a substantial influence on subunit combination. Removal of the hCG xcex2-subunit N-terminus led to loss in its ability to combine with the xcex1-subunit to form a heterodimer. This could be restored in part by changing the seatbelt residues of the xcex2-subunit to those found in the xcex2-subunit of hFSH. This suggested that interactions between different parts of the hormone subunits had significant roles in subunit combination. It also suggested that subunit combination was complex and that any modification of this region of the hCG xcex2-subunit might be expected to interfere with subunit combination. Indeed, work by Keutmann and colleagues (43) showed that synthetic peptides similar in structure to the N-Terminal region of the hCG xcex2-subunit inhibited subunit combination and that this portion of hCG was likely to be near the xcex1-subunit.
Sugahara et al. (44) showed that a fusion protein between the xcex1- and xcex2-subunits would lead to a protein that had many of the same properties as the heterodimeric parental molecule, including the ability to bind to receptors. Nonetheless, these analogs have all the amino acids of the protein connected together in a single-chain and therefore differ substantially from proteins that have two subunits. Unlike single chain proteins that are folded differently from the native hormones, hormone analogs that have two separate subunits similar to those found naturally would be expected to have receptor binding and immunological properties that are more similar to those of the parental molecules.